As recently reviewed by us, SERT-deficient +/- and -/- mice have gene-proportionate increases in extracellular fluid serotonin concentrations, i.e., 3- or 6-fold excesses respectively over wildtype +/+ mice. At the same time, SERT -/- mice have a marked deficit of intracellular, releasable serotonin. Despite increased extracellular levels of serotonin and decreased serotonin clearance, SERT +/- mice have unchanged tissue serotonin concentrations in the brain and periphery, and unchanged brain serotonin synthesis and turnover. Thus, a single copy of Slc6a4 is adequate to maintain overall serotonin tissue homeostasis. Continuing advances have been made in our studies of serotonin-related toxic reactions, including the serotonin syndrome. Most commonly, this toxicity occurs as a side effect in humans treated with certain antidepressant and anti-anxiety drugs. Importantly, its milder forms contribute to reduced therapeutic efficacy or a requirement to interrupt treatment in some individuals treated with SRIs. Our earlier studies exploring this behavioral and temperature-related syndrome in SERT-deficient mice revealed a genetic vulnerability to a markedly exaggerated serotonin syndrome when these mice were exposed to the metabolic precursor of serotonin, 5-HTP, or to other serotonergic drugs such as the monoamine oxidase-inhibiting (MAO-I) antidepressant tranylcypromine. Tranylcypromine is a clinically available antidepressant drug, and 5-HTP is readily available on internet sites and sold over-the-counter as a dietary supplement, and thus can be misused. We furthered these initial studies to show that SERT-deficient mice also have exaggerated behavioral responses to the atypical opioids tramadol and meperidine, prescribed as anti-pain medications. In addition to the serotonin syndrome behavioral changes, exaggerated alterations in temperature responses were also found in SERT- and MAO-deficient mice. We are now further extending these studies to include dopamine transporter (DAT) knockout mice. These findings in these mouse genetic models suggest the likelihood that humans with lower-expressing SLC6A4 SS genotypes, or other SERT or MAO variants that may lead to 50-80% decreases in SERT binding sites or transport function, may be at higher risk to develop serotonin syndrome neurotoxicity. This is based on highly congruent data from imaging, neuroendocrine, and other studies that have compared SERT-deficient mice, and less commonly SERT-deficient non-human primates, to humans with SERT and MAO gene variants. Of special note, it is likely that relatively mild serotonin syndrome occurrence may contribute to early discontinuation of SRIs and other side effects during SRI treatment that are strongly associated with the lower-expressing SLC6A4 SS/LgLg genotypes and S or Lg alleles, as well as other newly discovered lower-expressing variants in this gene, as reviewed this year and as described in our other report, MH000336-32 LCS. Given this transgenic mouse data and human SLC6A4 and MAO polymorphism data, we have formed an international collaborative effort - Genes Involved in SEroTonin Toxicity or GISETTO - in which we are examining functional variants in the multiple serotonin (SLC6A4, MAOA) and drug metabolizing gene groups (e.g., CYP2D6) in individuals developing serotonin toxicity and other serotonin-related toxicities, both retrospectively and prospectively. Understanding these genetic mechanisms will improve serotonergic drug safety and efficacy by identifying patients whose response to drugs might be genetically compromised, and thereby might be at risk of adverse and potentially life threatening drug reactions. SERT and MAO provide intriguing examples of likely mouse-human congruence in genetic vulnerability to serotonin toxicity features. Functional variants exist in additional genes that can also be postulated to confer vulnerability to serotonin neurotoxicity, for example, serotonin receptor genes. This past year, we extended our studies of the serotonin syndrome to mice with genetic deficiencies of monoamine oxidase (MAO). As predicted, we reported in a pharmacogenonics journal that these mice resemble to SERT-deficient mice in having exaggerated serotonin syndrome responses to pro-serotonergic agents. As humans have MAO polymorphisms that reduce the function of MAO, it is highly likely that these individuals would be a risk population for the serotonin syndrome during treatment with serotonergic medications. In addition to our examinations of the serotonin syndrome and related receptor mediation, we have discovered several other alterations in serotonin receptors in SERT-deficient mice. For example, SERT-deficient mice show abnormalities in serotonin 5-HT2C receptors that are expressed in amygdala, a brain region involved in anxiety and fear responses. Specifically in the three-chamber social interaction test, the 5-HT2C agonist mCPP decreased sociability and sniffing in SERT +/+ mice, indicative of the well-documented anxiogenic effect of this drug. This response was absent in SERT -/- mice. Likewise, in the open field test, the selective 5-HT2C agonist RO 60-0175 induced an anxiogenic response in SERT +/+ mice, but not in SERT -/- mice. Since 5-HT2C receptor pre-mRNA is adenosine-to-inosine edited, we evaluated the 5-HT2C receptor RNA editing profiles of SERT +/+ and SERT -/- mice in amygdala. Compared to SERT +/+ mice, SERT -/- mice showed a decrease in the less edited, highly functional 5-HT2C isoforms, and an increase in more edited isoforms with reduced signaling efficiency. This increased RNA editing could explain, at least in part, the decreased behavioral responses to 5-HT2C agonists in SERT -/- mice. These alterations in 5-HT2C receptor in amygdala may be relevant to humans with SERT and/or 5-HT2C polymorphisms who exhibit altered amygdala responses in brain imaging studies. In addition to 5HT2C receptor editing, we also studied an additional previously unrecognized regulator of gene expression related to microRNA functions. Most of our studies of SLC6A4 regulation have been of the 5 promoter region functional variants that we discovered including 5HTTLPR, rs25531 and rs25532 plus additional studies of the intronic variant STin2 in obsessive-compulsive disorder (OCD), Tourette Disorder (TD) and other disorders. In 2012, we published the first report showing that two microRNAs, mir-15a and mir-16, regulate SERT expression in human placental and rat brain raphe cells. Only mir-16 had previously been studied using indirect methods and only in mice. Overall, the data accumulated by our Lab, as referenced below and previously, support the use of different genetically modified mice as vulnerability models for humans with SERT, MAO and other gene variants with regard to gene-gene and gene-environment interactions that contribute to human diseases and their pharmacologic treatment.